Optimizing methods for human testicular tissue cryopreservation and spermatogonial stem cell isolation

Cryopreservation of testicular tissue before cancer therapy for fertility preservation in prepubertal boys with cancer is of great interest in reproductive medicine. Isolation of spermatogonial stem cells (SSCs) from cryopreserved tissues would be a suitable cell source to re-establish spermatogenesis after cancer therapy. We herein establish optimized protocols for cryopreservation of human testicular tissue and isolation of SSCs from cryopreserved tissue. We developed a freezing protocol that provided high testicular cell viability and supported structural integrity and tubular epithelium coherence similar to fresh tissue. Then, we established a protocol that allowed efficient isolation of functional SSCs from cryopreserved tissues. Isolated cells were found on the testicular basement membrane after xenotransplantation. Our results demonstrated the preservation of testicular tissue structure and high cell viability with efficient isolation of SSCs after testicular cryopreservation, which is promising for future therapeutic applications in fertility preservation.